The purpose of this pilot project is to investigate the enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK, HPPK, EC 2.7.6.3) as a target for intervention in disease caused by Mycobacterium tuberculosis (MTB). Unlike vertebrate cells, which acquire folates exogenously through active transport, MTB and many other bacteria must synthesize folate de novo. HPPK is an enzyme present early in the metabolic pathway for the synthesis of reduced folates from GTP. The absence of HPPK in the host makes this enzyme an attractive target for chemotherapy. Depletion of reduced folates through inhibition of this pathway leads to inhibition of DNA, RNA and protein synthesis. Comprehensive studies of the folate pathway in mycobacteria are lacking but genes coding for enzymes in the pathway have been identified through the Sanger Centre MTB genome sequencing project. A DNA sequence in the MTB genome database has been annotated as a probable folK coding for HPPK. For this pilot study, we propose to establish that the gene listed as Rv3606c codes for HPPK. Our objectives are to clone and express Rv3606c in Escherichia coil, and prove that the protein is functionally HPPK. We will also assess the essentiality of the gene by construction of HPPK-deficient MTB strains. This will be done in MTB by allelic exchange mutagenesis and a counterselection method based upon a mycobacterial thermosensitive origin of replication and toxicity of the sacB gene to MTB in the presence of sucrose. The results of this pilot study will enable us to better understand the biochemistry of folate metabolism in MTB. It will also provide purified HPPK for future drug discovery studies based upon structure-activity relationships, molecular modeling and crystallographic structure-based drug design.